Project Summary In order for the jaw to properly function, the jaw skeleton must be integrated to the underlying muscles through tendons. The prevalence of tendon injuries is high and in some cases can contribute to temporomandibular joint disorders, yet both the development and repair of tendons are largely understudied. The skeleton and tendons of the jaw are derived from cranial neural crest cells (CNCCs), in contrast to the skeleton and tendons of the fins/limbs and spine that are derived from mesoderm. Do tendons derived from different lineages depend on similar or distinct upstream signals for their specification? This proposal tests the innovative idea that tendon specification is modular, with head- and trunk-specific transcription factors initiating tendon development through head- and trunk-specific enhancers of critical tenocyte factors such as Scleraxis. By conducting single-cell RNA expression profiling of CNCC derivatives in the developing zebrafish face, we have found that expression of the nr5a2 orphan nuclear receptor marks cells along a developmental trajectory toward jaw tendon fate. Utilizing mutant and transgenic zebrafish models, we find that Nr5a2 is necessary and sufficient for specification of jaw tendon at the expense of cartilage fates. In addition, by assaying open chromatin indicative of active enhancers in CNCCs, as well as head versus trunk tenocytes, we find a number of putative head- and trunk-specific enhancers of scleraxis-a. In this proposal, we use transgenic and cutting- edge genomics techniques to test that Nr5a2 directly binds and activates jaw-specific scleraxis-a enhancers. We also use conditional genetics in mouse to test that Nr5a2 has a conserved role in specifying jaw and middle ear tendons derived from the mandibular arch in mammals. Completion of these aims will reveal the regulatory logic by which tendons are specified in different parts of the body, as well as a highly specific role for the Nr5a2 orphan nuclear receptor in promoting jaw tendon formation.